In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription

Abstract

Zombie active sites are only found in the cells were they are made, whereas the individual transcripts can diffuse away and be detected in cells other than their cell of origin. (a) Co-culture of mES-Z1 cells with non-transgenic parental cells. Active sites are only found in CFP+ cells. However, small diffraction limited dots are found in all cells including the non-transgenic ones. (b) Same image as a, but with blue and cyan channels turned off for better visibility of the barcode signal. A few examples of individual barcode transcripts are marked by circles. The inset shows a magnified and contrast adjusted view of a CFP negative cell, marked by the square, which contains some small barcode dots, indicating diffusion of individual RNA molecules from the cells in which they are produced. (c) In the absence of any transgenic cells, background non-specific signal is low. Indicating that the signal observed in the presence of transgenic cells is not non-specific HCR amplification. (d) Same image as c, but with blue and cyan channels turned off to make the lack of small dots more evident. The experiments were independently repeated three times with similar results. Scale bar is 25 μm. a b c d zBC Cerulean (mRNA) Cerulean (protein) DAPI zBC Cerulean (mRNA)

Imaging cell lineage with a synthetic digital recording system

“…Further, because the same recording design can work with additional integrases (Fig. S2), and recent work eliminates the need for in vivo transcription for FISH readout ( 61 ), it should be possible to build intMEMOIR systems with multiple recording ‘channels’ with different integrases regulated by specific signaling pathway or transcription factor activities ( 23 ). intMEMOIR thus provides a foundation for understanding how each cell’s present state emerges from its spatial context and individual history.…”

Application of combinatorial optimization strategies in synthetic biology

“…In this review, we discussed how DNA barcoding methodologies have significantly improved in recent years, enabling the computation of population dynamics 42,75 or tracking of mutants of Cas9 nuclease library 76 . Such techniques however have yet to be applied for tracking of combinatorial library members.…”

“…To do this, synthetic DNA cassettes with associated molecular barcodes were integrated into the E. coli genome, producing thousands of variants. Barcode sequences and microarrays were then used to compute population dynamics 42,75 . In another example, a pooled library of barcoded labelled mutants of Streptococcus pyogenes Cas9 (SpCas9) nuclease were easily tracked by high-throughput short-read sequencing 76 .…”

“…presented Zombie system for image-based readout of DNA barcodes 75 . In this system, phage RNA polymerases transcribe genomically integrated barcodes in fixed cells.…”

Encoding Method of Single-cell Spatial Transcriptomics Sequencing

“…The DNA microscope can encode the spatial location but it has limited targets and, so far, has not been widely used. There are numerous other advances in this field that are not described here in detail [33]. The development of these auxiliary tools will help us to understand and apply single-cell spatial transcriptomics.…”